Association between maternal smoke exposure and congenital heart defects from a case–control study in China

There is a gap in knowledge how maternal exposure to environmental tobacco smoke (ETS) is associated with offspring congenital heart defects (CHDs). In this case–control study, we collected data on 749 fetuses with CHDs and 880 fetuses without any congenital anomalies to examine the association of maternal ETS with fetal CHDs and the potentially moderating effect by maternal hazardous and noxious substances (HNS), periconceptional folate intake and paternal smoking. Maternal exposure to ETS in first trimester was associated with increased risk of CHDs in a dose–response gradient, with the AORs (95% CI) were1.38 (1.00–1.92), 1.60 (1.07–2.41), and 4.94 (2.43–10.05) for ETS < 1 h/day, 1–2 h/day, and ≥ 2 h/day, respectively. With the doubly unexposed group as reference categories, AORs for maternal ETS exposure ≥ 2 h/day in the absence of folate intake, in the presence of HNS exposure or paternal smoking, were 7.21, 11.43, and 8.83, respectively. Significant additive interaction between ETS exposure and maternal folate intake on CHDs was detected. Maternal ETS exposure during first trimester may increase the risk of offspring CHDs in a dose–response shape, and such effect may be modified by maternal folate intake or other potential factors.


Association between maternal ETS exposure and CHDs. As shown in
, maternal exposure to ETS from 12th month before pregnancy through first trimester was associated with an increased risk of CHD in offspring. The adjusted odds ratios (AOR (95% CI)) were 1.67 (1. 28 There seemed to be a statistically significant dose-response relationship between maternal ETS and offspring CHD (Table 3). We did not observe the association for ETS < 1 h per day, but found significant associations for higher dose of ETS exposure (1-2 h per day, AOR = 1.60, 95% CI 1.07-2.41; ≥ 2 h per day, AOR = 4.94, 95% CI 2.43-10.05).
Similar dose-dependent gradients were also found in the associations between ETS exposure and CHD subtypes. Nearly all the strong associations were found for exposure to ETS ≥ 2 h per day within each CHD subtype, such as the AOR (95% CI) of 4.87 (2.29-10.33) for SPD: 6.16 (2.44-15.53) for CTD, 4.63 (1.84-11.64) for LVOTO, 6.07 (2.54-14.53) for RVOTO, and 10.52 (3.65-30.29) for other CHDs. (Table 3). Table 4 presents the interactions of maternal ETS exposure (as four-level categorical variable) with maternal HNS exposure, and with paternal smoking (as binary variables). The highest AORs were observed for maternal ETS exposure ≥ 2 h per day in the absence of folate intake (AOR = 7.21, 95% CI 2.64-19.67), in the presence of HNS (AOR = 11.43, 95% CI 3.32-39.44), and in Table 1. Subject characteristics of the CHD cases and controls. a Taking folic acid over 90 days or more during the 3 months before conception to the first trimester. bMaternal HNS (maternal hazardous and noxious substances) exposure during the 3 months before conception to the first trimester. c Paternal smoking during 0-3 months before conception. www.nature.com/scientificreports/ congenital anomalies family history) were included in the models, the estimates for some indicators became no longer statistically significant, and some invalid estimates were generated.

Discussion
In this study, we observed that maternal ETS exposure in first trimester may increase the risk of CHD in the offspring, in a dose-response shape. In addition, the risks of CHDs or some subtypes were modified by intake of maternal folate supplement, HNS exposure and paternal smoking. Our findings are consistent with previous studies 8,9 . Tobacco smoke contains a large amount of toxins and carcinogens, including nicotine, carbon dioxide, carbon monoxide, carbonyls, etc. Nicotine and carbon monoxide both rapidly crosses the placenta, with chronic exposure, may contribute to fetal hypoxia 10 . Pregnant rats exposed to a mild concentration of carbon monoxide may delay postnatal electrophysiological maturation of ventricular myocytes from newborns rats, likely vulnerable to life-threatening arrhythmias 11 . Moreover, passive smoking contains elevated concentrations of harmful materials, perhaps even higher than for active smoking 12,13 . Both spermatozoa and the oocytes can be vulnerable to DNA damage due to tobacco smoke exposure 14,15 . Accumulating evidence demonstrate that maternal smoking before and after pregnancy as well as passive smoking may result in miscarriage, stillbirth, low birth weight, childhood asthma development and offspring CHDs [16][17][18][19] .
In our study, maternal exposure to ETS from 12th months before pregnancy through first trimester was associated with increased risk of CHDs in offspring, while such association was not for maternal ETS exposure during 0-12th months before pregnancy. Likewise, in a National Birth Defects Prevention Study, modest positive association were detected between maternal exposure to secondhand tobacco smoke during the period one month before conception through the first trimester and some CHD subtypes (conotruncal defects, aortic stenosis and atrial septal defects) 19 . In first trimester, the key period of fetal heart development and teratogenesis, fetal developing heart is vulnerable to hazardous and noxious stimuli resulting in cardiovascular anomalies 20 . Most CHDs occur in fetuses between 2 and 9 weeks of gestation, but many women are not aware of being pregnant during this period 21 . Fetal and neonatal health outcomes improves if mothers keeping away from the tobacco smoking or exposure early in pregnancy. www.nature.com/scientificreports/ Our study further provided evidence of the dose-response relationships between maternal ETS exposure during first trimester and CHDs. The dose-dependent associations of maternal active and passive smoking on the risk of CHDs were identified in previous studies 6,22,23 . By testing the hair nicotine concentration (HNC), Li et al. found 9 that compared with the low concentration group, the associations became sharply stronger in higher HNC on the risk of CHDs.
It has been generally recognized that periconceptional maternal folate intake may reduce the risk of CHDs 24,25 . In China, rural women can be offered folic acid freely if they plan to be pregnant since 'Folic acid for the prevention of neural tube defects' was launched in 2009. Although the proportion of women consuming folate intake supplements is increasing, many women did not take folate supplements during three months before and after conception. Over 50% of male adults smoke in China 26 , which contributes to high exposure of passive smoking, also for pregnant women. Moreover, due to the placental barrier is not completely impermeable to the passage of environmental harmful chemical contamination 27 , maternal HNS exposure during the first trimester represents the most critical window of exposure for CHDs [28][29][30] . In this study, both maternal not taking folic supplements and HNS exposure were identified as risk factors of CHDs. We obtained strong evidence of additive interactions between maternal folate intake and ETS exposure on CHDs and several subtypes, while the evidence of interaction between maternal HNS, paternal smoking and ETS exposures were relatively weak as such effects were only observed for some CHD subtypes in stratification analyses. The findings highlight the significance of maternal folic acid supplementation and staying away from toxic and harmful environment to reduce the risk of CHDs as well as other adverse outcomes in offspring.
Our study has several strengths. First, all CHDs, including stillbirth, aborted pregnancy and livebirths, were collected to minimize the potential selection bias. Second, all CHD cases were diagnosed by echocardiography test and excluded syndromic or chromosome abnormality. Livebirths with CHDs were followed up by one year after deliver to validate the diagnosis results. The classification of CHD subtypes was used into relatively homogeneous subgroups to present the underlying etiologies and the potential risk factors under study to maximize the potential for meaningful analysis. Detailed information about maternal ETS exposure and paternal and maternal smoking makes it possible to comprehensively estimate the effect of exposure on CHD. Third, the epidemiological characteristics were collected in first trimester of pregnancy so recall bias could be very minimal.
The study also has limitations. The study was conducted in one hospital only, so replication is needed. Still, West China Second University Hospital is a large and high quality hospital and each year over 15,000 deliveries are recorded. Maternal ETS and HNS exposure were based on self-report, without testing biomarker levels. Further researches are warranted to better understand the molecular basis of CHD as well as the influence of gene-environment interactions.

Conclusion
Maternal ETS exposure during first trimester may increase the risk of CHD in the offspring in a dose-response shape, and the risk maybe modified by maternal folate intake, HNS exposure, and paternal smoking. These findings highlight the importance of prescheduled pregnancy and prevention, including folic acid supplementation, paternal smoking cessation, maternal avoidance from ETS and HNS at home or workplace, etc. Preventive strategies to reduce the occurrence of major CHDs are necessary for those pregnant women or families exposed to ETS in the presence of other risk factors.

Materials and methods
Study population and data collection. This case-control study was performed at the West China Second University Hospital from January 2014 to December 2016. The hospital serves the entire south western part of China, and Tibet, and ranks in the top ten in both obstetrics and gynecology and pediatric in China 31 . Study inclusion criteria were singleton pregnancy, and for cases non-syndromic CHDs (Code Q20-26 by ICD-10), which referred to cases with CHDs only and without other non-cardiac anomalies, and for controls, no CHDs or other congenital malformations. Cases and controls with extra-cardiac abnormalities, syndromic diseases and chromosomal aberrations, or whose mothers were active smokers were excluded.
After providing signed informed consent, all participants were face-to-face interviewed in first, second, third trimester and one month after delivery 32 . The information of questionnaires included parental socioeconomic characteristics, congenital anomalies family history, maternal folic acid supplementation, exposure to ETS or HNS at home or workplace, etc.
The study was approved by the Ethics Committee of West China Second University Hospital and was based on the tenets of the Declaration of Helsinki.
Case classification. CHDs were diagnosed via prenatal systematically echocardiography. Livebirths were confirmed via routine examination, which included heart auscultation and a neonatal echocardiography within one week after birth. Stillbirths diagnosed with CHD were aborted according to standard process. Each CHD medical record was reviewed by specialists, neonatal cardiologists to confirm diagnostic results. All CHD cases were encoding according to International Classification of Diseases-10 (ICD-10).

Exposure measurements. Maternal Environmental Tobacco Smoke (ETS) was measured through mater-
nal self-report and was defined as exposure to tobacco smoke, at least an average of 15 min/day at home or workplace during 12rd before pregnancy to the first trimester 33 . There were three different exposure periods in: (a) 3rd-12th months before pregnancy, (b) 0-3rd months before pregnancy, and (c) the first trimester. We further created a categorical variable capturing duration of ETS exposure as (A) Equals interval (a) above; (B) Cumulative exposure from intervals (a) and (b); and (C) Cumulative exposure from intervals (a), (b) and (c) (Fig. 1). Maternal ETS exposure average dose during first trimester of pregnancy was classified into three groups: (1) less than 1 h per day; (2) 1-2 h per day; 3) 2 h or more per day. Maternal exposure to HNS during 3rd months before conception through the first trimester was measured through self-report, including lead, cadmium, mercury, oil paint, solder, pesticide, insecticide, formaldehyde or other chemical materials.
Maternal folate intake means mothers taking folic acid supplement over 90 days or more during the 3rd months before conception to the first trimester 32,34 . Statistical analysis. We estimated the association between maternal ETS and fetal CHDs, including doseresponse relationship, by calculating odds-ratios (OR) and two-sided 95% Wald-type confidence intervals from unconditional logistic regression 35 . First, we fitted a model only including a categorical covariate for maternal ETS and the nested categorical covariates for exposure interval and exposure dose. Second, we examined confounding by additionally adjusting for potential confounders, such as maternal age (< 25, 25-29, 30-34, and ≥ 35 years), maternal education level (≤ 9, 10-12, 13-16, and ≥ 17 school years), residence (urban vs rural), congenital anomalies family history (yes vs no), maternal folate intake (yes vs no), maternal HNS exposure (yes vs no), paternal smoking (yes vs no), if differed significantly between cases and controls examined by using Pearson Chi-square test. Third, we included parameters for interaction between maternal ETS exposure and each of covariates (maternal folate intake, maternal HNS exposure and paternal smoking), and assessed the individual and joint effects of the risk factors. Fourth, we evaluated the interactions between ETS and another risk factor (maternal folate intake, HNS exposure, and paternal smoking) on multiplicative scale, and additive scale as proposed by Knol and Vander Weele 36 . The relative excess risk due to interaction (RERI), attributable proportion due to interaction (AP), and synergy index (SI) with corresponding 95% CIs, were derived from the regression coefficients and covariance matrix from the multivariate logistic regression analyses by using an R function for additive interaction measures 37 , or the epiR and InteractionR package 38 . The 95% CI of the RERI or AP equal 0 and that of the SI equal 1 were defined as no additive interaction.
Last, we performed several sensitivity and subgroup analyses to assess the robustness of the interactive effects. (a) We compared the results of models which included ETS exposure as a multi-categorical variable with those of models including ETS as a binary variable. (b) To assess the effects of potential confounders, we adjusted for various covariates in the models (i.e., without any adjustment, adjusted for the two of three exposures when assessing the interactive effect of another one, fully adjusted by all the aforementioned covariates in models). (c) Subgroup analyses were performed across different groups stratified by maternal age (< 30 years vs ≥ 30 years), maternal education level (≤ 12 years vs > 12 years), residence (urban vs rural) and congenital anomalies family history (Yes vs No).